The following description provides a summary of information relevant to the present invention. It is not an admission that any of the information provided herein is prior art to the presently claimed invention, nor that any of the publications specifically or implicitly referenced are prior art to the invention.
The immunologic arts have advanced markedly over the past ten years. The complexity of the science explaining aspects of the field is immense. We set forth below in this section a discussion concerning known aspects of various elements involved in immunogenic responses and concepts in the art that are related to the invention disclosed herein.
T Cells
T lymphocytes (i.e., T cells) are part of the immune system, which defends the body against bacterial, viral and protozoal infection, as well as aberrant molecules that contain epitopes recognized as non-self. The recognition of non-self molecules as well as the destruction of infectious agents carrying non-self antigens is a function of T cells. These cells provide for the cell-mediated immune responses of adaptive immunity.
Infecting pathogens are generally accessible to extracellular antibodies found in the blood and the extracellular spaces. However, some infecting agents, and all viruses, replicate inside cells where they are not exposed to, and cannot be detected by, extracellular antibodies. In order for these foreign agents to be accessible to the cell-mediated immune response, the cells harboring such pathogens must either “express” antigenic motifs of the infecting agents on the surface of the cells or the antigenic motifs must be shed from-the-cells, e.g. by cell death, to be accessible to and subsequently expressed on the cell membrane of phagocytic antigen presenting cells (“APCs”) that participate in the immune process.
Antigens derived from replicating virus for example, are displayed on the surface of infected cells where they may be recognized by “cytotoxic” T cells which may then control the infection by recognizing the viral antigen and killing the cell. The actions of such cytotoxic T cells depends upon direct interaction between the antigenic motif of the infecting agent expressed on the surface of the infected cells and the T cell's receptors having a specificity for the motif.
Although T cells are important in the control of intracellular infections, some foreign agents evade such control because they replicate only in the vesicles of macrophages; an important example is Mycobacterium tuberculosis, the pathogen that causes tuberculosis. Whereas bacteria entering macrophages are usually destroyed in the lysosomes, which contain a variety of enzymes and bactericidal substances, infectious agents such as M. tuberculosis, survive because the vesicles they occupy cannot fuse with the lysosomes. The immune system provides for fighting such agents by a second type of T cell, known as a T helper cell, which helps to activate macrophages and induce the fusion of lysosomes with the vesicles containing the infecting agents. The helper cells also bring about the stimulation of other immune mechanisms of the phagocyte. T helper cells may further be involved in initiating and/or sustaining the immune system's release of soluble factors that attract macrophages and other professional APCs to the site of infection.
Additionally, specialized “helper” T cells play a central part in the destruction of extracellular pathogens by interacting with B cells. Depending on the type of infection being controlled, participating T helper cells may have an inflammatory or Th1-like phenotype, or a suppressive Th2-like phenotype.
T Cell Receptors
T cell receptors (TCRs) are closely related to antibody molecules in structure and are involved in antigen binding. Variability in the antigen binding site of the TCR is created in a fashion similar to antibodies in that a large capacity for diversity is available. The diversity is found in the CDR3 loops of TCR variable regions which are found in the center of the antigen-binding site of the TCR. The diversity that is obtainable by TCRs for specific antigens is also directly related to an MHC molecule on the APC's surface to which the antigenic motif is bound and presented to the TCR.
One type of MHC that is involved in presenting processed antigen is class II MHC. The antigen or peptide binding site for a peptide on a class II MHC molecule lies in a cleft between the alpha and beta chain helices of the MHC molecule. In another type of MHC, the class I MHC, the binding site for a peptide lies in a cleft between the two alpha helices of the alpha chain. From the arrangement of highly variable antigens complexing with MHC molecule alleles, it is understandable that the mechanism of TCR recognition involves a combined distribution of variability in the TCR which must correlate with a distribution of variability in the ligand (i.e., antigen/MHC molecule complex). (Garboczi, et al., Nature Vol. 384; 134–41; Ward and Quadri, Curr Op Immunol. Vol. 9:97–106; Garcia, et al., Science, Vol. 279:1166–72).
MHC Molecules
In general, T cell responses to non-self motifs depend on the interactions of the T cells with other cells containing proteins recognized as non-self. In the case of cytotoxic T cells and Th1 cells, non-self proteins (i.e. antigens) are recognized on the surface of the target cell (such as an infected cell). Th2 cells, on the other hand, recognize and interact with antigen presented by professional antigen presenting cells such as dendritic cells, and B cells. Dendritic cells non-specifically internalize antigen while B cells bind and internalize foreign antigens via their surface immunoglobulin. In any case, T cells recognize their targets by detecting non-self antigenic motifs (e.g., peptide fragments derived from for example, a bacterium or virus) that are expressed either on infected cells or other immune cells, e.g. phagocytic APC. The molecules that associate with these peptide or antigen fragments and present them to T cells are membrane glycoproteins encoded by a cluster of genes bearing the cumbersome name “major histocompatibility complex” (MHC). These glycoproteins were first identified in mice in studies examining the effects on the immune response to transplanted tissues. In humans, the MHC equivalent has been termed HLA for “human leukocyte antigen”. In general, the term MHC is used to describe generally the molecules in the mammalian immune system involved in the presentation of antigenic motifs to T cells. As used specifically in this Letters Patent, MHC means any major histocompatibility complex molecule, either class I or class II, from any mammalian organism including a human, such molecule comprising full-length MHC molecules or sub-units thereof further comprising MHC encoded antigen-presenting glycoproteins having the capacity to bind a peptide representing a fragment of an autoantigen or other non-antigenic or antigenic sequence (e.g., a peptide), said MHC further having an amino acid sequence that is expressed and purified from natural sources, or by any artificial means in prokaryotic or eukaryotic systems having different glycosilations, or of either natural or synthetic origin that contains or comprises a modification of a natural MHC sequence.
The actions of T cells depend on their ability to recognize antigenic motifs on cells (such as cells harboring pathogens or that have internalized pathogen-derived products). T cells recognize peptide fragments (e.g., pathogen-derived proteins) in the form of complexes between such peptides and MHC molecules that are expressed on the surface of “antigen presenting cells”.
The two types of MHC molecules, i.e., MHC class I and MHC class II, deliver peptides from different sources (class I being intracellular, and class II being extracellular) to the surface of the infected cell. The two classes of MHC molecules vary with respect to the length of peptides that they are able to present. The binding pocket of the MHC class I molecules is blocked at either end, thereby imposing severe restrictions on the size of peptides it can accommodate (8–10 residues). The binding groove of the MHC class II molecules on the other hand allows peptides to protrude from the ends, and consequently much longer peptides (8–30 residues) can bind. (Rudensky, et al. Nature, Vol. 353:622–27; Miyazaki, et al., Cell, Vol. 84:531–41; Zhong, et al., J. Exp. Med., Vol. 284:2061–66).
Antigen Processing
Peptides bound to MHC class I molecules are recognized by CD8+ T cells (cytotoxic T cells), and those bound to MHC class II molecules are recognized by CD4+ T cells (helper T cells). Two functional subsets of T cells are thereby activated to initiate the destruction of antigenic motifs, and thereby the source (e.g. a pathogen) which may reside in different cellular compartments. CD4+ T cells may also help to activate B cells that have internalized specific antigen, and in turn give rise to the stimulation of antibody production against the antigenic motifs of the extracellular pathogens.
Infectious or antigenic agents can reside in either of two distinct intracellular compartments. Viruses and certain bacteria replicate in the cytosol or in the contiguous nuclear compartment, while many pathogenic bacteria and some eukaryotic parasites replicate in the endosomes and lysosomes that form part of the vesicular system. The immune system has different strategies for eliminating such agents from these two sites. Cells containing viruses or bacteria located in the cytosol are eliminated by cytotoxic T cells which express the cell-surface molecule CD8. The function of CD8 T cells is to kill infected cells.
Immunogenic agents located in the vesicular compartments of cells (which may or may not have been involved in the internalization of extracellular matter) are detected by a different class of T cell, distinguished by surface expression of the molecule CD4. CD4 T cells are specialized to activate/modulate other cells and fall into two functional classes: Th1 cells which activate various immune competent cells to have the intravesicular non-self antigenic agents they harbor destroyed, and Th2 cells which help to activate B cells to, among other things, make antibody against such foreign agents.
To produce an appropriate response to infectious microorganisms, T cells need to be able to distinguish between self and foreign or non-self material coming from the different processing pathways. This is achieved through delivery of peptides to the cell surface from each of these intracellular compartments by the different classes of MHC molecules. As noted above, MHC class I molecules deliver peptides originating in the cytosol to the cell surface, where the antigen (i.e., non-self recognized peptide) is expressed in association with the MHC molecules (antigen:MHC complex) and is recognized by CD8 T cells. Likewise, MHC class II molecules deliver the non-self peptides originating from extracellular sources to the cell surface, where they are recognized by CD4 T cells.
Antigen Presenting Cells
When naïve T cells encounter for the first time a specific antigen on the surface of an antigen-presenting cell (APC), they are activated to proliferate and differentiate into cells capable of contributing to the removal of the antigen and its source (e.g. an infecting pathogen). The APCs are specialized in that they express surface molecules that synergize with a specific antigen in the activation of naïve T cells. APCs become concentrated in the peripheral lymphoid organs, to which they migrate after trapping antigen while circulating in the periphery. APCs present peptide fragments or antigenic motifs to recirculating naïve T cells. Arguably, the most important APCs are dendritic cells whose known function includes the presentation of antigen to Macrophages and are important in phagocytosis of cells that provide a first line of defense against infecting agents. APCs are also known to be activated by armed effector T cells. B cells also serve as APCs under some circumstances.
One of the features of APCs is the expression of co-stimulatory molecules including B7-1 and B7-2 molecules. Naïve T cells will respond to an antigenic motif only when the same APC presents to the T cell both the specific motif recognized by the TCR and a B7 molecule which is recognized by CD28 or CTLA-4, the receptors for B7 existing on the T cell surface. (Anderson, et al., J. Immunol., Vol. 159:4:1669–75). The activation of T cells by APCs leads to proliferation of the activated T cells and to the differentiation of their progeny into armed effector T cells. The proliferation and differentiation of T cells depends on the production of cytokines (such as the T cell growth factor, IL-2) and their binding to high-affinity receptors on the activated T cell. T cells whose TCRs are bound to antigens in the absence of co-stimulatory molecules fail to make cytokines and instead become anergic. This dual requirement for both receptor/antigenic interaction and co-stimulation helps to further mediate naïve T cell response.
Proliferating T cells develop into armed effector T cells, the critical event in most adaptive immune response. Once an expanded clone of T cells achieves effector function, the T cell clone progeny can act on any target cell that displays or expresses a specific antigen on its surface. Effector T cells can mediate a variety of functions. The killing of infected cells by CD8 cytotoxic T cells and the activation of professional APC by Th1 cells together make up cell-mediated immunity. The activation of B cells by both Th2 and Th1 cells help to produce different types of antibodies, thus driving the humoral immune response. (Kirberg, et al., J. Exp. Med., Vol. 186:8:1269–75).
T Cell Activation
T cells generally become sensitized to antigens by becoming trapped in lymphoid organs as the T cells drain into lymph nodes through which they circulate. Antigens introduced directly into the bloodstream, or that reach the bloodstream from an infected lymph node, are picked up by APCs in the spleen for example where lymphoid cell sensitization occurs in the splenic white pulp. The trapping of antigen by APCs that migrate to these lymphoid tissues combined with the continuous recirculation of T cells through the tissues ensures that rare antigen-specific T cells will encounter their specific antigen being presented by an APC.
The recirculation of naïve T cells through the lymphoid organs is orchestrated by adhesive interactions between lymphocytes and endothelial cells. Naïve T cells enter the lymphoid organs through a process which is thought to occur in a number of steps. The first step in this process is mediated by selectins expressed on the T cell. For example, L-selectin on naïve T cells binds to sulfated carbohydrates on the vascular addressins GlyCAM-1 and CD34. CD34 is expressed on endothelial cells in many tissues but is properly glycosylated for L-selectin binding only on the high endothelial venule cells of lymph nodes. L-selectin binding promotes a rolling interaction, which is critical to the selectivity of naïve lymphocyte homing. Although this interaction is too weak to promote extravasation, it is essential for the initiation of the stronger interactions that then follow between the T cell and the high endothelium, which are mediated by molecules with a relatively broad tissue distribution. (Finger, et al., Nature, Vol. 379:266–9).
Stimulation by locally bound chemokines activates the adhesion molecule LFA-1 on the T cell, increasing its affinity for ICAM-2, which is expressed constitutively on all endothelial cells, and ICAM-1, which, in the absence of inflammation, is expressed only on the high endothelial venule cells of peripheral lymphoid tissues. The binding of LFA-1 to 4ts4igands, ICAM-1 and ICAM-2 plays a major role in T cell adhesion to and migration through the wall of the blood vessel into the lymph nodes. Bachmann et al., Immunity, Vol. 7:549–57).
The high endothelial venules are located in the lymph nodes. This area is inhabited by dendritic cells, which have recently migrated from the periphery. The migrating T cells scan the surface of these APCs for specific antigen:MHC complexes. If they do not recognize antigen presented by these cells, they eventually leave the node via an efferent lymphatic vessel, which returns them to the blood so that they can recirculate through other lymph nodes. Rarely, a naïve T cell recognizes its specific antigen:MHC complex on the surface of an APC, which then signals the activation of LFA-1, causing the T cell to adhere strongly to the APC. Binding to the antigen:MHC complex also activates the cell to proliferate and differentiate, resulting in the production of armed, antigen-specific T cells. The number of T cells that interact with each APC in lymph nodes is very high, as can be seen by the rapid trapping of antigen-specific T cells in a single lymph node containing antigen.
Identification and Isolation of Antigen-Specific T Cells
As noted above, T cells represent a major component of the body's immune defenses against bacterial, viral and protozoal infections, as well as non-self antigenic motifs from other sources. T cells have also been implicated in the rejection of cancerous cells. Autoimmune disorders have also been linked to antigen-specific T cell attack against various parts of the body. One of the major problems hampering the understanding of and intervention on the mechanisms involved in these disorders is the difficulty in identifying T cells specific for the antigen to be studied. Accordingly, it is of great interest to be able to identify antigen-specific T cells. Additionally, it would be of great therapeutic benefit if T cells specific for a particular antigen could be (i) enriched and then reintroduced in a disease situation, (ii) selectively depleted in the case of an autoimmune disorder, or (iii) modified to alter their functional and/or phenotypic characteristics. Thus, identification and isolation of antigen specific T cells is an essential requirement in immunology and medicine to understand and modulate immune responses.
Identification of antigen-specific T cell populations is generally accomplished by indirect means in animal models, such as by evaluating membrane markers correlated to activation or maturation of these cells. The majority of these studies were performed in transgenic systems (Ignatowicz, et al., Cell, 84:521–29; Sebzda et al., Science, Vol. 263:1615–18; Jameson et al., Ann. Rev. Immunol., Vol. 13:93–126). Analysis is generally done by means of flow cytometry, where a detector on a machine is capable of identifying cells bound to fluorescent substrates, such as fluorescent antibodies. Positively identified cells can be sorted for further use. Quantitation and isolation of antigen-specific T cells is usually accomplished by limiting dilution and cloning techniques. When using sorted cells, these approaches become quite cumbersome and are sometimes inaccurate, since the biological effects of antigen recognition can spread beyond the cells recognizing the antigen. For instance, upon engagement of the specific MHC:antigenic peptide complex, T cells produce cytokines that can affect expression of the same markers of activation in non-specific bystander T cells. Hence, in order to isolate and characterize cells with specificity for a given antigen, alternative procedures, such as T cell cloning, need to be applied. These techniques often require many months of technical procedures before results can be obtained. The rate of success, in particular for human systems, is quite low, and the population selected may not necessarily represent the biologically relevant component of the immune response to a given peptide. The direct interaction of a specific T cell with the antigen:MHC complex would thus be a preferred basis for T cell isolation.
Theory of the Invention
The immunoregulation art has advanced steadily in recent years. The scientific literature contains many studies showing interactions and modulation effects between specific molecules and cell types. However, no discovery has been presented that is able to apply the knowledge that has been gained by the extensive research in the field toward a method or device that can be used in a comprehensive package for carrying out the identification, isolation, and modulation of immunoregulatory cells for the purpose of advancing the ultimate goal of such knowledge, i.e. improved treatment regimens for various states of disease.
We have discovered a platform technology for advancing treatment regimens requiring the immunoregulation of immune cells that centers around the use of an artificial antigen presenting cell (APC). This platform technology may be designed or programmed on demand for use in the treatment of a broad spectrum of specific disease states. Moreover, this system is versatile and applicable to all situations where the isolation, identification, and modulation of T cells is of clinical import. We have recognized the relevance of several types of molecular entities to the stimulation/activation and modulation response of T cells in their role within the immune system and have incorporated these entities into artificial APCs. We use such artificial APCs to capture and manipulate antigen specific T cells.
Historically, programming and using T cells therapeutically has been hampered by the problem of finding a means by which the cells can be handled for such manipulation and observation of the effectiveness of the manipulation applied. We have solved this problem by adopting the theory that a T cell can best be manipulated by using APC like structures and encorporating into such structures molecules constructed to (1) bind the “artificial” APC to specific T cell types, (2) stimulate or modulate only specifically bound T cells for any desired response, and (3) bind the artificial APC to a solid support in situations where anchoring the APC to a specific location is desired.
Prior to our invention, no comprehensive system has been disclosed, nor was it obvious that such a system would function as desired, to achieve a platform that is universally applicable to activating and modulation T cells. As can be seen by the numerous following distinctions, much of the art has centered only on basic research relating to molecules and their association with T cell response.
Distinctions
Kendrick et al., U.S. Pat. No. 5,595,881 (the '881 disclosure) discuss a method for the detection and isolation of MHC:antigen-restricted T cells which is performed by preparing the MHC:antigen complex, which complex is isolated by using metal chelating technology. The complexes then bound to a planar solid support {i.e., a glass coverslip), followed in turn by combining the immobilized complex with a biological sample so that the MHC:antigen complex may bind to and retain antigen-specific T cells. Determination of the presence of reactive MHC:antigen complexes is carried out by observation of cell proliferation.
The method described in the '881 disclosure differs from the current invention in a number of substantial structural and functional ways. First, the MHC component of the complexes in the '881 disclosure are immobilized on a solid support. The MHC component of the current invention is not bound to a solid support but is freely “floating” within the bilayer of a polysome membrane comprising a phosphotidylcholine and cholesterol component. The difference is substantial in that the MHC:antigen complex of the '881 disclosure is not able to participate in the migration or concentration of such complexes in “capping” which is important to improved binding and activation of bound T cells. Second, the '881 method is only directed to the detection of the presence of “natural” APCs that are specific for pre-selected antigen-specific T cells after such T cells have been isolated. The isolation of antigen-specific T cells is carried out by first performing a series of steps including binding antigen via a metal chelating process to a solid support, capturing on to the antigen MHC the components that are antigen-specific, then isolating the MHC:antigen complexes which are in turn bound to a planar solid support via a linker.
The current invention is also much more versatile. It is not concerned with detecting natural APCs but is instead directed to the isolation and manipulation of antigen-specific T cells. The manipulation of such T cells is carried out for numerous applications such as directly impacting T cell function by modulating the T cell response. The manipulation can be performed in either a column format, with means for supporting the artificial APCs, and/or in free solution via flow cytometry (FACS). The current invention is able to modulate T cell function because the artificial APCs may be designed to specification in that various functional molecules are incorporated into the APC that activate specific T cell responses. For example, in one embodiment of the current invention, known MHC molecules may be incorporated into liposomes along with a labeled antigenic peptide for which such MHC has specificity (e.g., in the case of FACS a biotinylated antigen). The liposome:MHC:biotinylated antigen complex may be used to bind to antigen-specific T cells and the fact of binding can be visualized by FACS followed by the sorting of the bound cells. Thus, no cell proliferation is necessary to identify and isolate antigen-specific T cells.
In addition to the MHC:antigen complex, the “artificial APCs” used to capture the antigen-specific T cells include accessory molecules to help stabilize the MHC:antigen:TCR interaction, and may also include functional molecules such as co-stimulatory molecules which in one embodiment may be used to activate T cells, adhesion molecules which may be used to bind cells destined for a certain area of the body, and other accessory or functional molecules such as cytokines or antibodies to cytokine receptors, which are known to have immunomodulatory effects upon T cells. Moreover, the current invention further provides for proper orientation of each of these molecules within the artificial APC membrane by a novel use of an anchoring mechanism comprising GM-1 and the β subunit of cholera toxin. In this aspect, the molecule of interest may be connected to the cholera toxin subunit as a fusion protein or by use of a linking moiety. By attaching the cholera toxin subunit to the molecule of interest, the cholera toxin may be bound by the GM-1 that is incorporated into and has affinity for the nonpolar region of the artificial APC membrane.
All of these molecules are incorporated into the liposomes of the artificial APCs in a free floating format. Other molecules may be included that do not influence the modulation of T cell response such as proteins that may be used to anchor the artificial APC to a solid support. Such molecules may also be produced as fusion proteins for proper orientation. As used herein such molecules that are not associated with modulation or T cell binding are termed “irrelevant” molecules.
Additionally, a label may be attached to the antigen, the irrelevant molecule, or the liposome component. Moreover, label may also be noncovalently associated within the lipids of the liposome.
The designs of these artificial APCs also allow for optional expansion experimentation of T cell populations responding to the MHC: antigen complexes associated in the cell like liposomes using a solution based (e.g., roller bottle) cell culture. The concept of the current invention represents a substantial and heretofore unrecognized advance in the MHC:antigen complex T cell binding art in that the artificial APC (e.g. the example comprising liposome:MHC:antigen:accessory molecule-functional molecule complex) is not restricted to complexes of MHC:antigen alone or to a planar surface as is the case with much of the prior art. The importance of the structural differences cannot be over emphasized. The addition of the accessory molecules, as well as co-stimulatory molecules, and other proteins in proper orientation in the liposomes of the current invention allow for substantially improved binding association and manipulation of T cells which is very important in the identification and stimulation of antigen-specific T cells. This is especially true in solution based FACS analysis where functionality of the antigen-specific T cells can be interpreted directly. For example, prior studies (Watts, T. H. Annals of the New York Academy of Sciences. 81:7564–7568.) respecting the modulation of T cells may be erroneous. There, it was demonstrated that planar membranes containing purified MHC loaded with antigen fused to glass cover slips elicited IL-2 production by T cells through the interaction of the T cell with the MHC:antigen complex. It was also shown that the same complex when formed in unilamellar vesicles (i.e., liposomes) elicited no response. Contrary to such teaching, we have found that liposome vesicles containing MHC: antigen complexes can in fact elicit strong response when combined with accessory molecules such as LFA-1, and other molecules such as co-stimulatory and adhesion molecules. We based our theory that liposomes could function without use of a planar array on the observation (by the same study cited immediately above) that crude membrane preparations of cellular material from which the MHC was purified were effective in eliciting T cell responses in both planar and vesicular forms. Subsequently, we have discovered that “extraneous” matter existing in cell extracts that might be hypothesized to impart functionality to vesicular forms of lipid bilayers (as opposed to unilamellar liposomes alone) are not important to T cell binding and response. Rather, T cell binding and response is possible using vesicular forms of liposomes containing specific molecules applied in combination with lipsomes (e.g., accessory molecules, co-stimulatory molecules, and adhesion molecules).
Prior research has also been inconclusive respecting the use of MHC molecules. For example, it has been shown (Buus, S. Cell. 47:1071–1077.) that a particular antigenic peptide binds solely to the alpha chain of the class II MHC IAd molecule while other investigations have shown that binding interactions between T cell receptors and MHC:antigen ternary complexes use whole MHC, not just single chains of the MHC, to determine peptide sequence motifs. Exactly how much of a MHC: antigen complex must be presented is not absolutely known and may vary with T cell specificity. We have directed our invention to the use of either whole MHC molecules or those parts of the a and β subunits of Class I and Class II MHC necessary for forming antigen binding cleft regions in the binding of antigen peptides.
The current invention's use of co-stimulatory, adhesion and other accessory molecules in a “free floating” format also helps to both anchor and direct the interaction between MHC:antigen:accessory molecule and T cell receptors by providing a means by which T cells in the sample will be presented with a structure more similar to that found in the natural state. Specifically, the MHC:antigen:accessory molecule complexes in conjunction with other functional molecules are able to migrate in proper orientation in the lipid bilayer of the liposome because of the use of a unique combination of lipids and surfactant molecules, namely an optimal ratio of phosphotidylcholine and cholesterol respectively, included in the liposome matrix. These provide particular protein presentation characteristics and easy protein migration properties to the surface of the liposome structure so that the MHC:antigen complexes can easily migrate to T cell binding loci similar to “capping” events seen in natural APCs. Moreover, as shown in the figures, the structure of our artificial APC liposomes allows for specific “capping” of the liposomes on the surface of the T cells to which the liposomes are bound. Additionally, interaction between the T cell and artificial APC-associated molecules is further enhanced by the molecules being oriented in the lipid membrane such that their active sites are positioned facing outward on the APC. Without such orientation, the ratio of properly oriented molecules to improperly oriented molecules is around 50:50. This ratio is greatly increased using MHC, functional and accessory proteins that have attached thereto (either by fusion protein construction or by use of a linker) a cholera β toxin subunit moiety which is placed in relation to the active center of the protein of interest such that upon the β subunit being bound by GM-1 which is incorporated into the lipid layer of the artificial APC, the protein of interest will lay in the APC with the active site facing outward.
Additional versatility is available with the current invention in that the artificial APCs may incorporate irrelevant molecules to be used in conjunction with separate solid support-based capture moieties for capturing generic target motifs such as irrelevant molecules. Because of the capacity for the functional molecules to migrate in the liposome, the irrelevant molecules may be nonspecifically directed away from the binding position of the T cells thus avoiding steric hindrances. Additionally, the system avoids a need for manufacturing specialized solid phase capture substrates for each antigen-specific complex.
With regard to the capture of the APC by the solid phase component of the invention, we refer to target molecules used in the artificial APC for binding to capture molecules of the solid support as “irrelevant” molecules because they do not impact the APC: T cell interaction. Such a design further preserves the ability of the other molecules inserted into the liposome to move freely and accommodate any capping of the T cell's activation related molecules.
It has been recognized that the number of receptors on a T cell is variable (Rothenberg, E. Science. 273:78–79.). It is also known that the number of TCRs and combination of co-stimulatory molecules and accessory molecules varies with the maturation of the T cell (Dubey, C. J. Immunol. 157:3820–3289.). How many such receptors are needed in all situations to elicit a T cell response is unknown. Moreover, it is known that presence of a co-stimulatory signal decreases the number of receptors necessary to activate a T cell (Viola, A. and Lanzavecchia, A. Science. 273:104–106.). We have provided for the uncertainties presented by such data by providing a system that allows control over the number of MHC:antigen:accessory molecule complexes relative to other functional molecules such as co-stimulatory, and adhesion molecules. The binding and modulation of the T cell response at different stages of cell maturation may be “fine tuned” using our invention.
In another system, Nag et al. in U.S. Pat. No. 5,734,023 (the '023 disclosure), disclosed MHC subunits which were complexed with antigenic peptides and “effector” molecules wherein such complexes were used to identify T cell populations that were associated with autoimmune diseases. The complexes were used to destroy and anergize such T cell populations from a patient's blood cell population.
The effector molecules are described as such things as toxins, radiolabels, etc. which may be conjugated to the MHC or antigen portion of the complexes and which may effecutate the identification, removal, anergy, or death of such T cell populations. Such effector molecules are not related to the attractive binding interactions or T cell responses to effectuate a phenotype change in the cells. They are merely designed and intended to aid in the recognition and/or destruction of specific T cell populations. Additionally, the '023 disclosure uses lipids in the construction of micelles which are designed for intravenous injection as therapeutics. The use of negatively charged acidic phospholipids (such as phosphatidylserine) and the lack of cholesterol or GM-1 and cholera toxin subunit in the design of such micelles differs from that of the current invention in substantial ways. For example, our invention uses neutrally charged phospholipids such as phosphotidylcholine (Pc). We have found that the design of our artificial APCs substantially increases stability because of the Pc and cholesterol in environments where IL-1 is present. IL-1 is known to interact with charged phospholipids and destabilize liposome structure. Likewise, in environments where TNF is present, the permeability of liposomes comprised of charged phospholipids (e.g., phosphotidylserine) is greatly affected. In the same manner, environments where RNase is present may also affect charged phospholipid liposome structures. We have avoided the disruptive effects caused by molecules that are often present in media from which T cells are isolated by designing artificial APCs using neutral phospholipids.
Additionally, the use of liposomes and the parameters associated with micelle construction that are disclosed in the '023 disclosure are wholly associated only with the stability of MHC:antigen:effector molecule complexes in the in vivo circulatory environment. There is no relation inherent or otherwise to the current invention, nor is there insight disclosed as to liposome construction containing co-stimulatory and adhesion molecules or protein orientation mechanisms such as the binding of cholera toxin by GM-1, or fused or linked moieties to the MHC, functional or accessory proteins of interest. Further, the '023 disclosure does not discuss use of its micelle construction in the context of use of a MHC: antigen complex ex vivo where manipulation of T cell function and the binding attraction between T cells and MHC: antigen complexes with respect to the current invention is of import. Moreover, the current invention does not use the technology disclosed in the '023 disclosure of single chain MHC in liposomes. In contrast, in a preferred embodiment, our invention uses either whole MHC molecules or those portions of the α and β subunits necessary to bind antigens and that may be designed to have substantially favorable liposome stabilizing characteristics as well as binding capabilities when in the presence of other functional molecules in the artificial APC as disclosed herein.
In yet another recent disclosure, Spack et al. in U.S. Pat. No. 5,750,356 (the '356 disclosure) describe a method for monitoring T cell reactivity using a modified ELISPOT assay which detects various factors produced by the stimulation of T cells with numerous factors in the presence of natural antigen presenting cells. The current invention is distinguishable from the '356 disclosed method in that the current invention uses artificial antigen presenting cells which have incorporated therein various accessory, co-stimulatory, adhesion, cytokine, and chemokine molecules that provide substantial effect in the binding and modulation of T cell responses. Additionally, in embodiments that require solid support binding, our APC includes irrelevant molecules. Moreover, in another embodiment, our invention includes mechanisms to properly orient proteins of interest in the lipid membrane.
In still another disclosure, Wilson et al. in U.S. Pat. No. 5,776,487 disclose a use of liposome structures for determining analyte in a test sample wherein the liposome contains only an analyte specific ligand and a haptenated component used to bind to a receptor moiety on a solid phase. This combination allows for capturing a test analyte onto a solid support for detection. Thus, it is vastly divergent from the concept of the current invention.
Our methods and artificial APCs are further distinguished from other recent disclosures. For example, Altman et al., in U.S. Pat. No. 5,635,363 (the '363 disclosure), discuss a method for labeling T cells according to the specificity of their antigen receptor by preparing a “stable multimeric complex” comprised of four or more MHC molecules having a substantially homogenous bound peptide population. The multimeric antigen:MHC complex was said to form a stable structure that because of its “stable multimeric” design, purportedly increases the affinity of a T cell receptor for its specific antigen thereby allowing for the labeling, identification and separation of T cells. Although such multimeric MHC components are known to bind T cells, they are not incorporated into liposome structures. Thus, the MHC complexes are unable to participate in capping type concentration. Moreover, the '363 method does not use accessory, co-stimulatory, adhesion, or other molecules to assist T cell binding and/or activation or modulation.
The current invention is further distinguishable over prior disclosures in that our invention is based on the recognition that the valency of the liposome: MHC structure is multiple, and empirically determined. Moreover, we have provided for greater specificity in following APC:T cell interaction due to one embodiment of our invention wherein the antigen is labeled rather than the MHC component (e.g. a biotinylated antigen with a streptavidin molecule conjugated to a fluorochrome).
In light of the above noted distinctions, the disclosed artificial APC and use of a separate solid support containing a binding protein to bind irrelevant molecules on the artificial APC represents an especially notable improvement over prior art. For example, in the above mentioned prior technologies the design of complexes are such that simultaneous binding and capping of the MHC: antigen and TCR/CD3/accessory molecules cannot occur. Capping is the phenomenon by which the T cell focuses the relevant molecules to the portion of the cell where binding has occurred, thus amplifying the binding, and subsequently the signaling of the event to the cell's other components. The current invention provides a specifically designed lipid bilayer similar to that of a natural cell which allows protein molecules, such as the MHC:antigen complexes to float freely, thus enabling the complexes to conform to any capping events the T cell may undergo. The consequence is a greater ability of the current invention to bind to, stimulate, and modulate T cells on demand.